International Conference on Environmental and Biological Sciences (ICEBS'2012) December 21-22, 2012 Bangkok (Thailand)

Fatty Acıd and Essentıal Oıl Composıtıon of Echınacea Purpurea (L.) Moench, Growıng in Kahramanmaras-Turkey

Emel Diraz1,Sengul Karaman 2, Nilufer Koca 3

proposed mechanism of action relating to its reported ability to Abstract— is a hardy perennial indigenous to stimulate to immune system [5].The immuno-stimulating effect North America, which belongs to or Compositae, have is caused by four reactions; activiating phagocytoses, medical properties. Echinacea purpurea (L). Moench cultivated in stimulating the fibroblast, increasing respiratory activity and Avşar Campus-Kahramanmaraş, gathered in Agust 2008. Seeds of increasing mobility of leucocytes [6]. According to the present the extracted for fatty acid composition with two different solvent (hexan and petroleumether) by using soxhalet apparatus, knowledge, genus Echinacea has identified seven groups of essential oils of the flowers were extracted by hydrodistillation. Fatty medically important components including polysaccaharides, acid and essential oil compositions were determined with GC-MS. flavonoids, caffeic acid derivates, essential oils, Principal fatty acid components with petroleumether and hexan polyacetylenes, alkylamides and miscellaneous chemicals [2] respectively, oleic acid % 48, % 29, palmitic acid % 16.6, % 9.2, which have been analysed applying various chromatographic linoleic acid % 13.3, % 51, stearic acid % 5, % 2.5. For essential oils germacrene-D % 11.3, caryophylleneoxide % 8.7, β-caryophyllene % separation techniques [7]. 7.2 α-cadinol % 6.3, naphthalene % 3.3, α-phellandrene % 2.9 were found main compounds. II. MATERIALS AND METHODS Essantiol oil and seed oil analyses determined with GC-MS. Keywords—Echinacea purpurea, fatty acid, essential oil, GC-MS analysis were conducted in the Plant Physiology GC-MS Laboratory in Biology Dept. of Kahramanmaras Sutcu Imam University. Qualification of the oil was analyzed on Agilent I. INTRODUCTION 5975C Mass Spectrometer coupled with Agilent GC-6890II

series. CHİNACEA is a hardy perennial plant indigenous to E North America, which belongs to Asteraceae or A. Essential Oil Analysis Compositae plant family and includes nine different species Dried plant material (20g Echinacea purpurea L. Mornch ) (Echinacea purpurea, Echinacea angustifolia, Echinacea was subjected to hydro-distillation method with 4h using a pallida, Echinacea atrorubens, Echinacea laevigata, Clevenger type apparatus for determining the oil content. The Echinacea paradoxa, Echinacea songuinea, Echinacea oil compositions were determined with GC-MS. The GC was simulata, Echinacea tenensseensis) [1]. Three species of equipped with HP-88 capillary column (100 m x 250 µm x Echinacea (Echinacea purpurea, Echinacea angustifolia, 0.20 µm film thickness) and He was used carrier gas with flow Echinacea pallida) have medical properties [2] and are rate of 1.0 mL/min. The GC oven temperature was o o o commercially traded as medicinal plants and used for programmed as follows: 70 C (1 min), 230 C at of 10 C/min and then kept at 230 oC at 20 min. The injector temperature floriculture. o Currently, extracts and whole plant products made from was 250 C. The mass spectrometer was operating in EI mode Echinacea purpurea, Echinacea angustifolia, Echinacea at 70 eV. Split ratio was 20:1. Mass range 35-400m/z; scan µ pallida comprise one of the largest sectors of the several speed (amu/s): 1000. 10 L of the oil was mixed 0.5 ml µ billion dolar herbal medicine market, in North America as well dichlora methan and 1 L of the concentrations injected into the column. The components of the oil were identified by as Europe. Annual sales of Echinacea products have been comparing their retention indices and mass spectra with those estimated $ 300 milion in the US alene [3]. İncreasing market of pure authentic samples and NIST98, Willey7n.1 and demand for Echinacea products has led to a rapid expantion of Flavor2 libraries reference compounds. Echinacea cultivation [4].Echinacea is used most widely as prevention or treatment for the commen cold, with the B. Seed Oil Analysis

Echinacea purpurea L. Mornch seeds 15 g were extracted Emel1. Dıraz1 Kahramanmaraş Sütçüimam University, Kahramanmaras, 46100, Turkey (phone: 00903442801400 ; fax: with petroleum ether (150 ml) and the other 15 g seeds were 00903442191042 ; e-mail: [email protected] . extracted with hexan (150 ml) for 6h using a soxhlet apparatus. The oil compositions were determined with GC-MS. The GC

35 International Conference on Environmental and Biological Sciences (ICEBS'2012) December 21-22, 2012 Bangkok (Thailand) was equipped with HP-88 capillary column (100 m x 250 µm x components with 57 % percentage. In the study of Agarwal et 0.20 µm film thickness) and He was used carrier gas with flow al. [10] the most abundant terpene found in E. Purpurea oil rate of 1.0 mL/min. The GC oven temperature was was germacrene-D, which showed a rise from 7.2 %- 33.5 % programmed as follows: 170 (1 min), 220 oC at of 10 oC/min June to December. In Hudaib et al. reseach [11] germacrene-D and then kept at 230 oC at 15 min. The injector temperature was found 57.8 % ratio. In the report of Vaverkova et al. [12] was 250 oC. The mass spectrometer was operating in EI mode germacrene-D comprises the 4.8 % of the total oil. In our at 70 eV. Split ratio was 20:1. Mass range 35-400m/z; scan study, germacrene-D (11.3 %) has the biggest peak area. Kan speed (amu/s): 1000. Echinacea purpurea L. Mornch seeds et al. [13] analysed two different Echinaceae species oil was (0.1g) vortexted with hexane (5ml) 5 min, then (Echinacea purpurea and Echinacea pallid) in Turkey under vortexed with 2N KOH (0.5ml) 5 min.and centrifuge 5 min. the organic farmer conditions and determined the essential oils 1μl of the concentrations injected into the column. The of these species and the major components in E. components of the oil were identified by comparing their purpurea were germacrene D (11.20%), spathulenol (8.83%), retention indices and mass spectra with those of pure authentic caryophyllene oxide (8.46%) and 1,5-epoxysalvial-4(14)-ene samples and wiley7n.1, Famdbwax.L, Famedb23.L, libraries (5.76%). Kan et al. and our findings are similar. reference compounds. TABLE I. III.RESULTS ESSENTIAL OILS OF FLOWERHEADS

peak Retention Component Area % time 1 11.662 β-pinene 1.3 2 12.002 α-phellandrene 2.9 3 13.357 p-cymene 2.6 4 16.046 β-elemene 2.1 5 16.674 β-caryophyllene 7.2 6 17.049 α-cadinene 1.0 7 17.43 Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7- 3.3 dimethyl-1-(1-ethylethyl)-,(1S-cis)- 8 17.729 Germacrene-D 11.3 9 19.733 α-farnasene 1.0 10 20.112 1,5 epoxysalvia-4(14)ene 3.3 11 21.44 Naphthalene, 1,2,3,4,4a,7-hexahydro-1,6- 1.6

Fig.1. Major Components Of Flowerheads Essential Oils dimethyl-4-(1-methylethyl)- 12 21.613 α-bisabolene 2.3

13 22.138 Bicyclo[4.4.0]dec-1-ene, 2-isopropyl-5- 1.8 The essential oil and fatty acids contents of Echinacea methyl-9methylene- purpurea were analyzed. The analyse of essential oils from the 14 22.346 Caryophyllene oxide 8.7 flowerheads was given in table I. The essential oil content of 15 22.613 γ- cadinene 2.0 E. purpurea flowerheads was 1.5 % (w/v). The essential oil 16 22.962 α-cadinol 6.3 was separeted to 89 components, 63 of them were identified, 17 23.267 Decatone 1.5 comprising 92.8% of the total yield (Table I). 18 23.434 Ethyl oleate 1.6 19 23.653 Vulgarol. B 1.9 The major components of the E. Purpurea are germacrene- 20 23.916 Aromadendrene oxide 1.1 D (11.3%), caryophyllene oxide (8.7 %), β caryophyllene (7.2 21 24.596 3,4-difloro-4 methoxybiphenyl 2.7 %), α-cadinol (6.3 %). The essential oil content was 22 24.867 Isoaromadendieneepoxide 1.9 monoterpene hydrocarbons, oxygenated monoterpens, 23 25.306 Trans (Z)- α-bisabolene epoxide 2.3 sesquiterpens and oxtgenated sesquiterpens. 5.4 % of the E. 24 26.072 Benzenepropanoic acid, octadecyl ester 1.2 25 26.104 Diepi-.alpha.-cedrene epoxide 1.2 Purpurea oil belongs to monoterpene hydrocarbons such as α- Those lower than 1% were excluded. pinene, camphane, β-pinene, myrcene, limonene, p-cymene, thujone. β-caryophyllene, α-cadinene, germacrene-D, α- farnesene, α-bisabolene, isospathulenol, were sesquiterpene hydrocarbons comprised 22.8 %, caryophyllene oxide, aromadendrene oxide and isospathulenol were oxygenated sesquiterpens comprised 10.4 % of the total oil content. Myrtenol was an oxygenated monoterpene found % 0.2 ratio. In earlier research by Mazi and Cottrell [8] E. Purpurea, E. Angustifolia and E. Pallida roots, stems, leaves and flowers oils were analyzed. Terponoids of head space of flowers and stems were about 81 %- 91%. In the study by Salehi et al.[9] oxygenated monoterpene was not found in the E. Purpurea oil and 6.4 % monoterpene hydrocarbons, 70.9 % sesquiterpene Fig. II Most Abundant Fatty Acids In Seeds With Different Solvents hydrocarbons and 15.4 % oxygenated sesquiterpens were found. Germacrene-D was the highest ratio among the

36 International Conference on Environmental and Biological Sciences (ICEBS'2012) December 21-22, 2012 Bangkok (Thailand)

TABLE II. of the plant. FATTY ACIDS OF SEEDS Echinacea has shown good adaptation to Mediterranian region. pe minute component Area % ak Petroleum n-hexane REFERENCES ether 1 10.766 Oxocol-C13 0.1 - [1] R.L., McGregor, “The of the genus Echinacea (Compositae)”, 2 10.911 Myristic acid-C14:0 0.3 0.2 The University of Kansas Sci. Bull. 48, 113-142; Lancaster, pp. 45-73, 1968 3 11.585 Pentadecanoic acid-C15:0 0.1 0.1 [2] R. Cozzolinoa, P. Malvagnaa, E. Spinaa, A. Giorib, N. Fuzzatib, A. 4 12.369 Palmitic acid-C16:0 16.6 9.2 Anellib, D. Garozzoa, G. Impallomenia,, “ Structural analysis of the 5 12.990 Palmitoleic acid-C16:1 1 0.4 polysaccharides from Echinacea angustifolia radix,” Carbohydrate 6 13.227 Carbomic acid-C17:0 0.2 0.1 Polymers, Vol. 65( 3), pp 263–272, August 2006. 7 13.893 Heptadecenoic acidC17:1 0.2 - [3] P. Brevoort , “The US botanical market: a new overview” Herbalgram, 8 14.210 Stearic acid-C18:0 5 2.5 vol. 44, pp. 33–48, 1998 . 9 14.673 Elaidic acid-C18:1 0.7 - [4] Y. Chen, T. Fu, İ. Tao , J. Yang ,Y. Chang, ‡ M. Wang , L. Kim, L. Qu,, 10 14.972 Oleic acid-C18:1 48 29 J. Cassady, R. Scalzo, X. Wang, “Macrophage Activating Effects of New 11 15.383 Linolelaidic acid-C18:2 0.8 - Alkamides from the Roots of Echinacea Species.” Nat. Prod., vol. 68 (5), 12 15.701 Linoleic acid-C18:2 13.3 51 pp 773–776, April 2005. 13 16.515 Arachidic acid-C20:0 0.5 0.6 [5] K Bone, D Phyto, “Echinacea: When should it be used,” Alt Med 14 16.816 Gamma-linolenic acid-C18-3 - 0.5 Rev, 2(6), pp. 451-458, 1997. 15 17.406 Eicosenoic acid-C20:1 0.3 0.5 [6] L. Thygesenb, J. Thulinb, A. Mortensena, L. H. Skibsteda, P. Molgaardb, 16 19.500 Behenic acid-C22:0 1.3 0.7 “ Antioxidant activity of cichoric acid and alkamides from Echinacea 17 21.333 Tricosanoic acid-C23:0 0.1 0.1 purpurea, alone and in combination.”Food Chemistry, Vol. 101(1), pp. 18 23.165 Lignoceric acid-C24:0 0.6 0.3 74–81 , 2007, 19 25.811 Oxiraneoctanoic acid-C26 0.8 0.8 [7] H. Schulz, S. Pfeffer, R. Quilitzsch, B. Steuer, K. Reif, “Rapid and Non- Destructive Determination of the Echinacoside Content in Echinacea Roots by ATR-IR and NIR Spectroscopy”, Planta Med 68(10), pp. 926- 929, 2002. Echinacea purpurea seed oil was extracted two different [8] Mazza G and Cottrell T., “Volatile components of roots, stems, leaves and solvent (n-hexan, petroleum ether). There are some differences flowers of Echinacea species.” J. Agric. Food. Chem. vol. 47, pp. 3081– in result of the fatty acids proportion. In the seed oil 18 3085,1999. [9] M. H., Mirjalili, P., Salehi, H. N. Badi, A. Sonboli, “Volatile constituents components were identified, comprising 90.4 % of total area of the flowerheads of three Echinacea species cultivated in Iran.” Flavour with solvent petroleum ether and 15 fatty acids determined and Fragrance Journal . vol. 21, 355–358, 2006. with solvent n-hexan, comprising 96 %. The most abundant [10] R. K. Thappa, S. K. Bakshi, P. L. Dhar, S.G. Agarwal, S. Kitchlu, M.K. Kaul, K.A. Suri, “Significance of changed climatic factors on essential fatty acids were palmitic acid, stearic acid, oleic acid, linoleic oil composition of Echinacea purpurea under subtropical conditions.” acid. While oleic acid 48 % , palmitic acid 16.6 %, linoleic Flavour Fragr. J., vol.19, pp. 452–454, 2004 acid 13.3 % with petroleum ether, oleic acid 29 %, palmitic [11] M. Hudaib, V. Cavrini, M.G. Bellardi, C.R. Autonell ,”Characterization of the esential oils of healthy and virus infected Echinacea purpurea (L.) acid 9.2 %, linoleic acid 51 % with solvent n-hexane. Oxocol, Moench plants.” J. Essent. Oil Res. vol.14, pp. 427–430, 2002. heptadecenoic acid, elaidic acid, linolelaidic acid could not [12] S. Vaverkova, M. Mikulasova, M. Haban, M. Holla, P. Otepka, defined with solvent n-hexane. Oomah et al.[14] research the “Variability of the essential oil from three sorts of Echinacea MOENCH fatty acids of E. angustifolia, E. Pallida, E. Purpurea and the genus during ontogenesis.” Ceska Slov. Farm., Vol.56 (3), pp.121-4, 2007. [13] Y Kan, M Kartal, US Ucan, AR Gulpinar, R major fatty acids of E. Purpurea with solvent hexan were Kan,”Antimicrobial activity and composition of the essential oils of linoleic (66.5%), oleic (21.4%), and palmitic acids (8.0%) in Echinacea species cultivated under organic farming conditions in the year 1998 harvest and linoleic (75.6%), oleic (12.2%), Turkey.” Planta Med, vol.74, pp. I55, 2008. [14] B. D. Oomah, D. Dumon, A. Cardador-Martı´nez, D. V. Godfrey, palmitic acids (7.0%) in the harvest 1999. Vandyshev et al. “Characteristics of Echinacea seed oil.” Food Chemistry, vol.96, pp. [15] extracted the fatty oils from the fruits of Echinacea 304–312, 2006 purpurea and E. pallida and major components were found [15] V. V. Vandyshev, E. Y. Babaeva, D. D. Drozdovskaya,” Triacylglycerols of the lipid fraction from fruits of linoleic (58.2%) and oleic (20.2%) acids. Major components two Echinacea species.”Pharmaceutical Chemistry Journal, Vol. 43, No. are the same with our findings but percentage of the 3, pp. 32 – 34, March, 2009. components showed differences.

IV.CONCLUSION

The fatty acid profile of Echinacea seed oil was similar to literatures. Oil composition of Echinacea purpurea extracts from the different solvents had some different chemical compositions. Solvents affected the percentage of components. Petroleum ether is more convenient solvent for extraction seeds oil. Echinacea purpurea flowerheads had high essential oil contents from literature. This results may be caused by the environmental conditions specially climatic conditions, development stage, harvest time, extraction method and origin

37 International Conference on Nanotechnology and Chemical Engineering (ICNCS'2012) December 21-22, 2012 Bangkok (Thailand)

Response of Biochemical Activities on Selected

Rice Plants towards Ozone Stress

M.Ismail, A.Suroto is firstly caused by reduction in the number of hectares /acres for rice growing areas [9]. Most of these areas have been Abstract-Superoxide dismutase (SODs) and glutathione converted for development purposes. Thus, production of reductase (GR) are two important enzymes as they are the first line rice is targeted to come from existing cultivated areas. of defence system within a plant cell against abiotic stress. Air pollutants and in particular tropospheric ozone is one of the abiotic However with the air quality that getting worse in Malaysia stresses that could give adverse effects on rice, the most dominant day by day; this situation also contributes to the reduction of food crop in Malaysia. Studies conducted had found that reduction rice production from the existing plant. This is because on SODs and GR activities will cause declining on production of studies have been conducted found that between air rice. Therefore in this study, response of SODs and GR activities on pollutants (i.e PM10, NOx, SOx, CO2, and ozone), they found three local rice cultivar i.e MR263, MR219 and MR84 towards that ozone had give significant impact on rice. The ozone (120 parts per billion, ppb) was conducted. Results show that there is significant different on SODs and GR activities between significant impact could be seen when there exist reduction control and exposed plants and also among exposed plants in production rice in the range of 10%-20% [4]-[6],[10]- (P<0.05). Moreover, MR84 (old cultivar) was found to be the most [11]. This scenario happened when biochemical activities tolerant cultivar compared to newer varieties. especially SOD’s and GR activities as the first parts in plant was affected by ozone and then influenced on production of Keywords--Superoxide dismutase (SOD), glutathione reductase rice [1, 2, 3]. Therefore in this study, there are three varieties (GR), tropospheric ozone, rice plants of local rice cultivar were selected i.e MR263 (newer cultivar), MR219 (current cultivar) and MR84 (old cultivar). I. INTRODUCTION SOD’s and GR activities for each cultivar will be identified UPEROXIDE dismutase (SOD) and glutathione between non-exposed and exposed plants also between Sreductase (GR) are important enzymes due to the fact exposed plants. that their function as the first defence system within a plant II. MATERIAL AND METHODOLOGY cell. They are able to fight and remove the oxidants in the cell parts involved [1]-[3]. Oxidant i.e hydrogen peroxide This study was conducted in Universiti Malaysia (H2O2) was identified come from air pollutants especially Terengganu (5.94ºN; 103.42ºE) in Fig.1. All the plants were ozone. Ozone or tropospheric ozone is one of the abiotic planted in a plot by using transplanted technique suggested stresses which a secondary pollutant that resulting from by Department of Agricultural (DOA) Malaysia. All seeds reaction between two main substances i.e nitrogen oxides were soaked for 48 hours and then evaporated for 48 hours. (NOx) and volatile organic compound (VOCs). These two They were cultivated for 15 days and after that period; they main substances come from natural sources and human were transferred into the bucket. There are 30 buckets for activities. Mostly NOx and VOCs have been reported come each variety and in each bucket, three seeds were planted. from human activities such as industrialization, urbanization Fourteen days before the panicle Initiation (P.I), six buckets and transportation [4]-[8]. Reaction between these two of each variety were randomly selected to be exposed to substances has formed ozone which nowadays becomes a ozone at concentrations of 120 part per billion (ppb). For threat towards rice. Therefore rice was selected in this study ozone exposure, there are two closed-top-chambers (CTCs) due to its importance as a major staple food in Malaysia. as in Fig.2 were used which both are identical and use Majority of people make rice as daily diet as well as their mechanical ventilation system i.e air conditioners for main source of carbohydrates. Demands on rice increased regulating temperature and relative humidity. They are only 3% by year and based on that scenario, it could be seen that different when one was supplied with the ozone and another production of rice is very important. However, rice one become as control (no additional ozone). Exposure to production is still lacking because of the constraints which ozone was conducted for 3 consecutive days, 8 hours/day. Day after exposure, 0.1g fresh sample was taken and M.Ismail is with Department of Engineering Science, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21300 Kuala Terengganu, enzyme activities superoxide dismutase (SOD) was Malaysia.(corresponding author: [email protected], measured using spectrophotometer at 540nm wavelength Tel.: +6096683548 Fax.: 09-6694660), based on the method proposed by Fridovich [12]. A.Suroto is with Department of Engineering Science, Faculty of Science and A unit of SOD is defined as the amount of enzyme that Technology, Universiti Malaysia Terengganu, 21300 Kuala Terengganu, Malaysia. (email: [email protected]) inhibited the rate of nitroblue tetrazolium (NBT) reduction This study was supported by Ministry of Agricultural (MOA) Malaysia by 50%. For determination of GR activity, 0.15g fresh grant 05-01-SF1011. sample was taken and enzyme activities were measured at 340 nm wavelength based on the method by Carlberg and

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